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Reactive oxygen species (ROS) function as key signaling molecules to inhibit stomatal opening and promote stomatal closure in response to diverse environmental stresses. However, how guard cells maintain basal intracellular ROS levels is not yet known. This study aimed to determine the role of autophagy in the maintenance of basal ROS levels in guard cells. We isolated the Arabidopsis autophagy-related 2 (atg2) mutant, which is impaired in stomatal opening in response to light and low CO2 concentrations. Disruption of other autophagy genes, including ATG5, ATG7, ATG10, and ATG12, also caused similar stomatal defects. The atg mutants constitutively accumulated high levels of ROS in guard cells, and antioxidants such as ascorbate and glutathione rescued ROS accumulation and stomatal opening. Furthermore, the atg mutations increased the number and aggregation of peroxisomes in guard cells, and these peroxisomes exhibited reduced activity of the ROS scavenger catalase and elevated hydrogen peroxide (H2O2) as visualized using the peroxisome-targeted H2O2 sensor HyPer. Moreover, such ROS accumulation decreased by the application of 2-hydroxy-3-butynoate, an inhibitor of peroxisomal H2O2-producing glycolate oxidase. Our results showed that autophagy controls guard cell ROS homeostasis by eliminating oxidized peroxisomes, thereby allowing stomatal opening. [ABSTRACT FROM AUTHOR]

Stomata regulate gas exchange between plants and atmosphere by integrating opening and closing signals. Stomata open in response to low CO2 concentrations to maximize photosynthesis in the light; however, the mechanisms that coordinate photosynthesis and stomatal conductance have yet to be identified. Here we identify and characterize CBC1/2 (CONVERGENCE OF BLUE LIGHT (BL) AND CO2 1/2), two kinases that link BL, a major component of photosynthetically active radiation (PAR), and the signals from low concentrations of CO2 in guard cells. CBC1/CBC2 redundantly stimulate stomatal opening by inhibition of S-type anion channels in response to both BL and low concentrations of CO2. CBC1/CBC2 function in the signaling pathways of phototropins and HT1 (HIGH LEAF TEMPERATURE 1). CBC1/CBC2 interact with and are phosphorylated by HT1. We propose that CBCs regulate stomatal aperture by integrating signals from BL and CO2 and act as the convergence site for signals from BL and low CO2. [ABSTRACT FROM AUTHOR]

Stomata within the plant epidermis regulate CO2 uptake for photosynthesis and water loss through transpiration. Stomatal opening in Arabidopsis thaliana is determined by various factors, including blue light as a signal and multiple phytohormones. Plasma membrane transporters, including H+-ATPase, K+ channels and anion channels in guard cells, mediate these processes, and the activities and expression levels of these components determine stomatal aperture. However, the regulatory mechanisms involved in these processes are not fully understood. In this study, we used infrared thermography to isolate a mutant defective in stomatal opening in response to light. The causativemutation was identified as an allele of the brassinosteroid (BR) biosynthetic mutant dwarf5. Guard cells from this mutant exhibited normal H+- ATPase activity in response to blue light, but showed reduced K+ accumulation and inward-rectifying K+ (Kin+) channel activity as a consequence of decreased expression of major Kin+ channel genes. Consistent with these results, another BR biosynthetic mutant, det2-1, and a BR receptor mutant, bri1-6, exhibited reduced blue light-dependent stomatal opening. Furthermore, application of BR to the hydroponic culture medium completely restored stomatal opening in dwarf5 and det2-1 but not in bri1-6. However, application of BR to the epidermis of dwarf5 did not restore stomatal response. From these results, we conclude that endogenous BR acts in a long-term manner and is required in guard cells with the ability to open stomata in response to light, probably through regulation of Kin+ channel activity. [ABSTRACT FROM AUTHOR]

In green plants, the blue light receptor kinase phototropin mediates various photomovements and developmental responses, such as phototropism, chloroplast photorelocation movements (accumulation and avoidance), stomatal opening, and leaf flattening, which facilitate photosynthesis. In Arabidopsis, two phototropins (phot1 and phot2) redundantly mediate these responses. Two phototropininteracting proteins, NONPHOTOTROPIC HYPOCOTYL 3 (NPH3) and ROOT PHOTOTROPISM 2 (RPT2), which belong to the NPH3/RPT2- like (NRL) family of BTB (broad complex, tramtrack, and bric à brac) domain proteins, mediate phototropism and leaf flattening. However, the roles of NRL proteins in chloroplast photorelocation movement remain to be determined. Here, we show that another phototropininteracting NRL protein, NRL PROTEIN FOR CHLOROPLASTMOVEMENT 1 (NCH1), and RPT2 redundantly mediate the chloroplast accumulation response but not the avoidance response. NPH3, RPT2, and NCH1 are not involved in the chloroplast avoidance response or stomatal opening. In the liverwort Marchantia polymorpha, the NCH1 ortholog, MpNCH1, is essential for the chloroplast accumulation response but not the avoidance response, indicating that the regulation of the phototropin-mediated chloroplast accumulation response by RPT2/NCH1 is conserved in land plants. Thus, the NRL protein combination could determine the specificity of diverse phototropinmediated responses. [ABSTRACT FROM AUTHOR]

Phototropins are light-activated receptor kinases that mediate a wide range of blue light responses responsible for the optimization of photosynthesis. Despite the physiological importance of phototropins, it is still unclear how they transduce light signals into physiological responses. Here, we succeeded in reproducing a primary step of phototropin signaling in vitro using a physiological substrate of phototropin, the BLUS1 (BLUE LIGHT SIGNALING1) kinase of guard cells. When PHOT1 and BLUS1 were expressed in Escherichia coli and the resulting recombinant proteins were incubated with ATP, white and blue light induced phosphorylation of BLUS1 but red light and darkness did not. Site-directed mutagenesis of PHOT1 and BLUS1 revealed that the phosphorylation was catalyzed by phot1 kinase. Similar to stomatal blue light responses, the BLUS1 phosphorylation depended on the fluence rate of blue light and was inhibited by protein kinase inhibitors, K-252a and staurosporine. In contrast to the result in vivo, BLUS1 was not dephosphorylated in vitro, suggesting the involvement of a protein phosphatase in the response in vivo. phot1 with a Cterminal kinase domain but devoid of the N-terminal domain, constitutively phosphorylated BLUS1 without blue light, indicating that the N-terminal domain has an autoinhibitory action and prevents substrate phosphorylation. The results provide the first reconstitution of a primary step of phototropin signaling and a clue for understanding the molecular nature of this process. [ABSTRACT FROM AUTHOR]

Light is a major environmental factor required for stomatal opening. Blue light (BL) induces stomatal opening in higher plants as a signal under the photosynthetic active radiation. The stomatal BL response is not present in the fern species of Polypodiopsida. The acquisition of a stomatal BL response might provide competitive advantages in both the uptake of CO2 and prevention of water loss with the ability to rapidly open and close stomata. We surveyed the stomatal opening in response to strong red light (RL) and weak BL under the RL with gas exchange technique in a diverse selection of plant species from euphyllophytes, including spermatophytes and monilophytes, to lycophytes. We showed the presence of RL-induced stomatal opening in most of these species and found that the BL responses operated in all euphyllophytes except Polypodiopsida. We also confirmed that the stomatal opening in lycophytes, the early vascular plants, is driven by plasma membrane proton-translocating adenosine triphosphatase and K+ accumulation in guard cells, which is the same mechanism operating in stomata of angiosperms. These results suggest that the early vascular plants respond to both RL and BL and actively regulate stomatal aperture. We also found three plant species that absolutely require BL for both stomatal opening and photosynthetic CO2 fixation, including a gymnosperm, C. revoluta, and the ferns Equisetum hyemale and Psilotum nudum. [ABSTRACT FROM AUTHOR]

PP1 (protein phosphatase 1) is among the most conserved enzymes known, with one or more isoforms present in all sequenced eukaryotic genomes. PP1 dephosphorylates specific serine/threonine phosphoproteins as defined by associated regulatory or targeting subunits. In the present study we performed a PP1-binding screen to find putative PP1 interactors in Arabidopsis thaliana and uncovered a homologue of the ancient PP1 interactor, I-2 (inhibitor-2). Bioinformatic analysis revealed remarkable conservation of three regions of plant I-2 that play key roles in binding to PP1 and regulating its function. The sequence-related properties of plant I-2 were compared across eukaryotes, indicating a lack of I-2 in some species and the emergence points from key motifs during the evolution of this ancient regulator. Biochemical characterization of AtI-2 (Arabidopsis I-2) revealed its ability to inhibit all plant PP1 isoforms and inhibitory dependence requiring the primary interaction motif known as RVXF. Arabidopsis I-2 was shown to be a phosphoprotein in vivo that was enriched in the nucleus. TAP (tandem affinity purification)-tag experiments with plant I-2 showed in vivo association with several Arabidopsis PP1 isoforms and identified other potential I-2 binding proteins. [ABSTRACT FROM AUTHOR]

Recent genetic analysis showed that phototropins (phot1 and phot2) function as blue light receptors in stomatal opening of Arabidopsis thaliana, but no biochemical evidence was provided for this. We prepared a large quantity of guard cell protoplasts from Arabidopsis. The immunological method indicated that phot1 was present in guard cell protoplasts from the wild-type plant and the phot2 mutant, that phot2 was present in those from the wild-type plant and the phot1 mutant, and that neither phot1 nor phot2 was present in those from the phot1 phot2 double mutant. However, the same amounts of plasma membrane H+-ATPase were found in all of these plants. H+ pumping was induced by blue light in isolated guard cell protoplasts from the wild type, from the single mutants of phototropins (phot1-5 and phot2-1), and from the zeaxanthin-less mutant (npq1-2), but not from the phot1 phot2 double mutant. Moreover, increased ATP hydrolysis and the binding of 14–3-3 protein to the H+-ATPase were found in response to blue light in guard cell protoplasts from the wild type, but not from the phot1 phot2 double mutant. These results indicate that phot1 and phot2 mediate blue light-dependent activation of the plasma membrane H+-ATPase and illustrate that Arabidopsis guard cell protoplasts can be useful for biochemical analysis of stomatal functions. We determined isogenes of the plasma membrane H+-ATPase and found the expression of all isogenes of functional plasma membrane H+-ATPases (AHA1-11) in guard cell protoplasts. [ABSTRACT FROM AUTHOR]

The epidermis of Argenteum mutant of Pisum sativum L. and Vicia faba L. was shown to be effective in protecting mesophyll photosynthesis from UV‐C irradiation (peak 254 nm; 1.5 W m−2). These plants were chosen because it is easy to peel the epidermis from both sides of the leaf in Argenteum and the abaxial side in Vicia. Chlorophyll a fluorescence induction was decreased to the same extent by UV‐C radiation in both leaf sides when the epidermis was removed. The fluorescence from the leaf with the epidermis inact was not affected. With irradiation of leaves with higher intensity (3.4 W m−2) of UV‐C, the variable fluorescence was decreased by UV‐C impinging on the abaxial side, but not the adaxial side in either of the plant species. A methanol extract from the mutant Pisum epidermis had a high absorbance in the UV region, and the absorbance was more than two‐fold larger in adaxial epidermis than in abaxial epidermis. These results indicate that the epidermal layer, which contains substances that have high UV absorbance, protects mesophyll photosynthesis against UV radiation. [ABSTRACT FROM AUTHOR]

Phototropins (phot1 and phot2) are blue light (BL) receptors that mediate responses including phototropism, chloroplast movement and stomatal opening, and increased cytosolic Ca2+. BL absorbed by phototropins activates plasma membrane H+-ATPase in guard cells, resulting in membrane hyperpolarization, and drives K+ uptake and stomatal opening. However, it is unclear whether the phototropin-mediated Ca2+ increase activates the H+-ATPase. Here, we determined cytosolic Ca2+ concentrations in guard cell protoplasts (GCPs) from Arabidopsis transformed with aequorin. Cytosolic Ca2+ increased rapidly in response to BL in GCPs from both the wild type and phot1 phot2 double mutants, but was mostly suppressed by an inhibitor of photosynthetic electron flow (DCMU). With depleted external K+, we observed another slower Ca2+ increase, which was phototropin- dependent. Fusicoccin, a H+-ATPase activator, mimicked the effect of BL. The slow Ca2+ increase thus appears to result from membrane hyperpolarization. The slow Ca2+ increase was suppressed by external K+ and was restored by blockers of inward-rectifying K+ channels, CsCl and tetraethylammonium, suggesting the preferential uptake of K+ over Ca2+. Such efficient K+ uptake in response to BL was not found in mesophyll cells. Both the fast and the slow Ca2+ increases were inhibited by Ca2+ channel blockers (CoCl2 and LaCl3) and a chelating agent (EGTA). These results indicate that the phototropin-mediated Ca2+ increase was not observed prior to H+-ATPase activation in guard cells and that Ca2+ entered guard cells via Ca2+ channels through photosynthesis and phototropin-mediated membrane hyperpolarization. [ABSTRACT FROM AUTHOR]

Sphingoid long-chain base (LCB) 1-phosphates are degradated by LCB 1-phosphate lyase to C16 fatty aldehydes and phosphoethanolamine. Here, we confirmed that the At1g27980 gene product, AtDPL1, is a functional LCB-1-phosphate lyase. Expression of green fluorescent protein fusion products in suspension-cultured Arabidopsis cells showed that AtDPL1 is located to the endoplasmic reticulum. The rates of fresh weight decreases of dpl1-1 and dpl1-2 mutants were significantly slower than those of the wild-type plants. This ability to limit their transpiration reflected the leaf temperature of the mutant plants more than that of wild-type plants, suggesting that AtDPL1 plays a role in dehydration stress. [ABSTRACT FROM AUTHOR]

Under drought stress, ABA promotes stomatal closure to prevent water loss. Although protein phosphorylation plays an important role in ABA signaling, little is known about these processes at the biochemical level. In this study, we searched for substrates of protein kinases in ABA signaling through the binding of a 14-3-3 protein to phosphorylated proteins using Vicia guard cell protoplasts. ABA induced binding of a 14-3-3 protein to proteins with molecular masses of 61, 43 and 39 kDa, with the most remarkable signal for the 61 kDa protein. The ABA-induced binding to the 61 kDa protein occurred only in guard cells, and reached a maximum within 3 min at 1 μM ABA. The 61 kDa protein localized in the cytosol. ABA induced the binding of endogenous vf14-3-3a to the 61 kDa protein in guard cells. Autophosphorylation of ABA-activated protein kinase (AAPK), which mediates anion channel activation, and ABA-induced phosphorylation of the 61 kDa protein showed similar time courses and similar sensitivities to the protein kinase inhibitor K-252a. AAPK elicits the binding of the 14-3-3 protein to the 61 kDa protein in vitro when AAPK in guard cells was activated by ABA. The phosphorylation of the 61 kDa protein by ABA was not affected by the NADPH oxidase inhibitor, H2O2, W-7 or EGTA. From these results, we conclude that the 61 kDa protein may be a substrate for AAPK and that the 61 kDa protein is located upstream of H2O2 and Ca2+, or on Ca2+-independent signaling pathways in guard cells. [ABSTRACT FROM AUTHOR]

Recent evidence suggests that nitric oxide (NO) acts as an intermediate of ABA signal transduction for stomatal closure. However, NOs effect on stomatal opening is poorly understood even though both opening and closing activities determine stomatal aperture. Here we show that NO inhibits stomatal opening specific to blue light, thereby stimulating stomatal closure. NO inhibited blue light-specific stomatal opening but not red light-induced opening. NO inhibited both blue light-induced H+ pumping and H+-ATPase phosphorylation. The NO scavenger 2-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) restored all these inhibitory effects. ABA and hydrogen peroxide (H2O2) inhibited all of these blue light-specific responses in a manner similar to NO. c-PTIO partially restored the ABA-induced inhibition of all of these opening responses but did not restore inhibition of the responses by H2O2. ABA, H2O2 and NO had slight inhibitory effects on the phosphorylation of phototropins, which are blue light receptors in guard cells. NO inhibited neither fusicoccin-induced H+ pumping in guard cells nor H+ transport by H+-ATPase in the isolated membranes. From these results, we conclude that both NO and H2O2 inhibit blue light-induced activation of H+-ATPase by inhibiting the component(s) between phototropins and H+-ATPase in guard cells and stimulate stomatal closure by ABA. [ABSTRACT FROM AUTHOR]